This proposal will develop analytical methodology to identify key metabolic pathways and the metabolizing enzymes of botanicals responsible for drug interactions. Initially we will focus on the main active ingredients of widely used botanicals, green tea (epigallocatechin gallate), St. John's wort (hypericin and hyperforin), and milk thistle weed (silybin). Metabolism of these compounds will be investigated in vitro in liver microsomes and expressed human enzymes. Liquid chromatography coupled with tandem mass spectrometry will be used to identify the phase I metabolites. The cytochrome P450 isoforms will be identified using chemical inhibitor probes and specific expressed enzymes. Enzyme kinetic and inhibition parameters of these compounds will be evaluated to predict the interactions with known drugs. A web-accessible, interactive relational database will be developed to include the findings such as specific isoforms, metabolites formed, metabolic reactions and enzyme kinetics and the inhibition constants of the above standards. The database will be expanded in Phase II to also include in vivo data of botanical extracts and their synergistic effects to provide information to pharmacists and physicians. PROPOSED COMMERCIAL APPLICATION: The procedures and automated methods developed in these studies will be used to provide contract research services to pharmaceutical/botanical industry. The research will result is a database that could be sold to healthcare professionals and pharmaceutical industry.